Journal of Cell Science 105, 1115-1120 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
1115
The BiP protein and the endoplasmic reticulum of Schizosaccharomyces
pombe: fate of the nuclear envelope during cell division
Alison L. Pidoux* and John Armstrong†
Membrane Molecular Biology Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln’s Inn Fields, London
WC2A 3PX, UK
*Present address: Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720, USA
for correspondence at present address: School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
†Author
SUMMARY
A polyclonal antibody was raised to the C-terminal
region of fission yeast BiP. The use of this antibody for
immunoprecipitation, western blotting and immunofluorescence has confirmed and extended the observations
made previously with an epitope-tagged BiP molecule.
A fraction of BiP protein is glycosylated in Schizosac charomyces pombe cells. Pulse-chase experiments
showed that this modification occurs rapidly upon synthesis and that the extent of glycosylation does not then
change with time. BiP protein is induced by elevated
temperatures and by treatment with tunicamycin. The
antibody cross-reacts with proteins of similar molecular weight in the yeasts Kluyveromyces lactis and
Schizosaccharomyces japonicus. Immunofluorescence of
BiP has been used to follow the behaviour of the ER
and in particular the nuclear envelope through the cell
cycle.
INTRODUCTION
genetic analysis, but differs from that organism in many
aspects of its molecular and cell biology. We have previously described the cloning and analysis of the BiP gene
from S. pombe (Pidoux and Armstrong, 1992). Like its
homologue in S. cerevisiae, it is essential for viability and
its mRNA is induced by various forms of stress. Unlike
BiP proteins from most other species, however, the
encoded protein contained a predicted site for N-linked
glycosylation. By overexpressing an altered form of the
gene encoding an immunological ‘tag’, we presented evidence that this glycosylation site was used, but only in a
small proportion of the molecules. The same tagged BiP
protein was used to visualise the ER of S. pombe by
immunofluorescence, revealing the nuclear envelope as
well as a peripheral reticulum reminiscent of that found
in higher cells. These observations, however, had the
potential limitation of requiring the expression of an
altered protein at abnormal and variable levels in the population of cells.
We describe here the direct observation of BiP in wildtype S. pombe, using an antibody raised to part of the protein expressed in bacteria. We have compared the alterations in protein levels under different forms of stress to
those of the corresponding mRNA, and monitored the efficiency and kinetics of glycosylation. The antibody is shown
to recognise an appropriately sized protein in two other
yeast species. The immunofluorescence analysis of the ER
of S. pombe has been extended to show the behaviour of
the nuclear envelope during the cell cycle.
The luminal compartment of the endoplasmic reticulum
(ER) contains a subset of proteins that are soluble but nevertheless are not secreted by vesicle transport out of the
cell. One member of this group is BiP, a protein originally
identified by its presence in the ER of pre-B cells, bound
to heavy-chain immunoglobulin in the absence of light
chains (Haas and Wabl, 1983). The protein is now known
to be ubiquitous, and is a member of the hsp70 family of
stress-regulated proteins (Munro and Pelham, 1986). Unlike
other members of this family, BiP is synthesised with a
cleavable N-terminal signal sequence for translocation into
the ER, and a C-terminal sequence specifying its localisation within the ER (Munro and Pelham, 1986, 1987). BiP
appears to function in regulating the folding and assembly
of a variety of membrane and secretory proteins. This
process requires binding to the nascent protein followed by
release coupled to ATP hydrolysis; in the cases of some
mutant proteins, the binding is irreversible (reviewed by
Gething and Sambrook, 1992). In the budding yeast Sac charomyces cerevisiae the BiP protein, which is encoded
by the gene KAR2, is additionally required for translocation of nascent proteins across the ER membrane, as well
as in an undefined role in fusion of nuclei after mating
(Rose et al., 1989; Normington et al., 1989; Vogel et al.,
1990).
The fission yeast Schizosaccharomyces pombe is a
simple eukaryote that, like S. cerevisiae, is amenable to
Key words: hsp70, glycosylation, cytokinesis
1116 A. L. Pidoux and J. Armstrong
MATERIALS AND METHODS
Yeast strains
S. pombe strain 972 and Schizosaccharomyces japonicus were
from Prof. Jeremy Hyams, University College, London;
Kluyveromyces lactis and Pichia pastoris from Dr Kevin Hardwick, Laboratory for Molecular Biology, Cambridge.
Preparation of anti-BiP antibody
A 400 bp HindIII fragment from the 3′ region of the BiP gene
(Pidoux and Armstrong, 1992) was inserted into the vector pATH3
for production of trpE-BiP fusion protein (Spindler et al., 1984).
Escherichia coli containing the pATH3-BiP plasmid were grown
overnight at 37°C in 5 ml M9CA medium containing 20 mg/ml
tryptophan, 30 µg/ml ampicillin, diluted into 50 ml of the same
medium without tryptophan and grown at 30°C with vigorous aeration for 1 h. Then, 250 µl of 1 mg/ml indole acrylic acid was
added and the incubation continued for 3 h. Cells were pelleted
and resuspended in 1 ml 10 mM NaPO4, pH 7.2, 1% β-mercaptoethanol, 1% SDS, 6 M urea and incubated at 37°C for 2-3 h.
An equal volume of 2× sample buffer was added and the sample
heated to 95°C for 5 min before loading onto a 10% SDS-PAGE
gel. Proteins were transferred from the gel to nitrocellulose membrane by semi-dry blotting and visualised by Ponceau S staining.
The trpE-BiP fusion protein band was excised from the filter,
washed, dried and dissolved in dimethyl sulphoxide.
For production of glutathione-S-transferase-BiP fusion protein,
the vector pGEX-2T (Smith and Johnson, 1988) was first digested
with BamHI and EcoRI and a synthetic polylinker was inserted.
The resulting plasmid, pGEX20T, contained adjacent restriction
sites for BamHI, EcoRI, XhoI, ClaI, SpeI and XbaI, while the original EcoRI site was destroyed. An EcoRI-XhoI fragment from the
3′ end of the BiP gene (Pidoux and Armstrong, 1992) was then
inserted. A saturated 100 ml culture of E. coli containing the
pGEX-20T-BiP plasmid was inoculated into 1 l of L-broth containing ampicillin and incubated for 1 h. Fusion protein was
induced by addition of IPTG to 100 mM and the culture grown
for a further 5 hours. Cells were pelleted and resuspended in 15
ml ice-cold 1% (v/v) Triton X-100 in phosphate-buffered saline
(PBS-TX) and placed on ice. Cells were lysed by sonication (several 3 s bursts at full power). Then 2 ml of pre-swollen glutathioneagarose beads (Sigma) in PBS-TX was added to the lysate and
incubated for 5 min at room temperature with inversion. Beads
were pelleted by centrifugation at 1700 r.p.m. for 2 s and washed
5 times with ice-cold PBS-TX. The beads were gently transferred
to a 2 ml tube and washed 3 times. Fusion protein was eluted by
washing three times with 1 ml 0.5 M glutathione (Sigma), 50 mM
Tris-HCl, pH 7.0, for 2 min at room temperature, and dialysed
against PBS.
A rabbit was injected with 0.5 mg of trpE fusion protein in
complete Freund’s adjuvant. After several boosts of 0.5 mg of
fusion protein in incomplete Freund’s adjuvant at 4-week intervals, no antibodies reactive against BiP had appeared. Therefore
the same rabbit was immunised by a similar protocol with the glutathione-S-transferase fusion protein.
Methods for induction of BiP, endoglycosidase H digestion,
35S-labelling of cells and immunoprecipitation, electrophoresis,
fluorography and western blotting, were as before (Pidoux and
Armstrong, 1992), with the exception that protein extracts were
prepared by disruption with glass beads. Yeast cells in log phase
were pelleted, resuspended in 2× sample buffer, an equal volume
of acid-washed glass beads (425-600 µm; Sigma) was added and
the cells were disrupted by vigorous vortexing. Extracts were
immediately heated to 95°C for 5 min, centrifuged briefly and the
supernatants taken for gel electrophoresis. Efficient transfer of
proteins to membranes for western blotting was confirmed by
staining with Ponceau S before immuno-labelling. Glutathione-Stransferase-ypt5 fusion protein (Armstrong et al., 1993) was provided by Dr S. Ponnambalam. To demonstrate specificity of the
antibody, each fusion protein was added during western blotting
at a concentration of 1 µg/ml. Conventional and confocal immunofluorescence were as described (Pidoux and Armstrong, 1992)
except that cells were fixed by addition of 1/10 volume 37%
formaldehyde to growing cultures and incubation continued at
30°C for 30 min. The anti-BiP antibody was used at a dilution of
1:100 for immunoprecipitation, 1:20000 for western blotting, and
1:100 for immunofluorescence. Protein A-Sepharose was used
directly for immunoprecipitation (Pidoux and Armstrong, 1992),
while peroxidase-conjugated and fluorescein-conjugated goat antirabbit antibodies (both Tago) were used for western blotting and
immunofluorescence, at dilutions of 1:1000 and 1:100, respectively.
RESULTS
Production and characterisation of anti-BiP
antibodies
The C-terminal 110 to 130 amino acids of S. pombe BiP
were used to make fusion proteins for immunisation of a
rabbit. This region of the protein was chosen because it
shows minimal homology to other members of the hsp70
family. Serum was tested for cross-reactivity against S.
pombe proteins by western blotting (Fig. 1). Two species
of 75-80 kDa were detected by the antibody in wild-type
cells (lane 2), but not by pre-immune serum (lane 1). Binding of the antibody could be inhibited by incubation with
glutathione-S-transferase-BiP fusion protein (lane 3) and
was specific to the BiP portion of the fusion protein since
incubation with glutathione-S-transferase-ypt5 fusion pro-
92 kDa
69 kDa
Fig. 1. Detection of S. pombe BiP protein by western blotting
(lanes 1-4) and immunoprecipitation (lane 5). A doublet of the
predicted size is recognised by immune (lane 2) but not preimmune (lane 1) serum. Binding is prevented by BiP fusion
protein (lane 3) but not by ypt5 fusion protein (lane 4). GST,
glutathione-S-transferase.
BiP from fission yeast 1117
Fig. 3. Induction of BiP protein by heat
shock and tunicamycin treatment. Equal
numbers of cells were incubated at 30°C
(lane 1) or 39°C for 30 min (lane 2) or
with 1 µg/ml tunicamycin at 30°C for 2
h (lane 3). In each case cells were
labelled for the last 30 min of
incubation, and the BiP protein
immunoprecipitated. Both treatments
cause some induction of protein; in the
presence of tunicamycin only the
unglycosylated form is observed.
Tm, tunicamycin
Fig. 2. Partial glycosylation of S. pombe BiP. The upper band of
the BiP doublet detected by western blotting (lane 1) is removed
by digestion with endoglycosidase H (lane 2). Pulse-chase
analysis of 35S-labelled protein (lanes 3-7) shows no change in the
proportion of the glycosylated form in protein immunoprecipitated
after 5, 10, 30, 60 and 120 min after the start of the chase.
tein showed no such effect (lane 4). The anti-serum could
also immunoprecipitate both BiP species from metabolically labelled cells (lane 5). Therefore the antiserum appears
to be specific for S. pombe BiP protein.
The BiP protein
S. pombe BiP contains a single potential N-linked glycosylation site near the N terminus of the protein. We have
previously shown that a fraction of epitope-tagged BiP
expressed from a plasmid receives core carbohydrate modifications (Pidoux and Armstrong, 1992). To confirm that
this phenomenon was not restricted to the altered version
of the BiP protein, S. pombe protein extracts were incubated overnight in the presence or absence of endoglycosidase H and analysed by western blotting (Fig. 2, lanes 1
and 2). The upper band disappears upon endoglycosidase
H treatment, indicating that it contains N-linked carbohydrate. A pulse-chase experiment was performed to investigate whether glycosylation occurs immediately upon synthesis or gradually, with all BiP molecules eventually
becoming glycosylated. After 5 min of labelling, immunoprecipitated BiP was present as both the glycosylated and
unglycosylated forms (Fig. 2, lane 3), with the glycosylated
form accounting for approximately 10% of the total proA
tein. The proportion of glycosylated protein did not change
throughout the chase period up to 2 h (lanes 4-7). These
observations indicate that BiP is glycosylated concomitantly with or very shortly after its synthesis.
In mammalian cells BiP is induced by agents such as
tunicamycin and calcium ionophores but not by heat shock.
S. pombe BiP mRNA is induced by heat shock and by treatment with tunicamycin (Pidoux and Armstrong, 1992). To
determine whether BiP protein levels were also affected by
these treatments, BiP was immunoprecipitated from
labelled cells. Cells subjected to 30 min heat shock at 39oC
contain a higher level of the glycosylated and unglycosylated forms of BiP (Fig. 3, lane 2) than untreated cells (lane
1). In cells treated with 1 µg/ml tunicamycin for 2 h (lane
3) only the unglycosylated form is present, as expected for
an inhibitor of N-linked glycosylation. The level of the unglycosylated form is higher in these cells than in the
untreated control.
Cross-reactivity of anti-BiP antibody
Although BiP proteins from different species show low
homology in their C-terminal regions, it was possible that
the antibody raised against S. pombe BiP would cross-react
with other yeast BiP proteins and might therefore be useful
for their study. Protein extracts were made from
Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia
pastoris, Schizosaccharomyces japonicus and S. pombe
cells, and analysed by western blotting using the anti-BiP
antibody (Fig. 4A). Cross-reactive species of the expected
B
92 kDa
69 kDa
Fig. 4. (A) Reaction of the antiserum with other yeast species. Equivalent amounts of extracts from S. pombe (lane 1), K. lactis (lane 2), S.
japonicus (lane 3), S. cerevisiae (lane 4) and P. pastoris (lane 5) were analysed by western blotting with the BiP antibody. K. lactis and S.
japonicus have a faint cross-reacting band of the appropriate mobility. (B) Alignment of the C-terminal regions of BiP protein sequences
from K. lactis (K. l.) and S. pombe (S. p.), showing regions of consensus (con).
1118 A. L. Pidoux and J. Armstrong
Fig. 5. Immunofluorescence of BiP.
(A) Conventional epifluorescence
microscopy shows labelling of the
nuclear envelope and elements close
to the plasma membrane.
(B,C) Confocal microscopy. A
section through the centre of cells
(B) shows similar structures to (A).
An image from approximately
2 µm above the centre of the cells (C)
reveals in addition a network of
peripheral tubules. Bars: 10 µm (A);
2 µm (B, C).
mobility for BiP were detected in K. lactis and S. japonicus
extracts, though the bands were fainter than for S. pombe
BiP. S. japonicus and S. pombe are related fission yeasts. In
contrast, the budding yeast K. lactis , whose BiP gene has
been cloned (Lewis and Pelham, 1990), is much more distantly related to S. pombe. However, a comparison of the
C-terminal amino acid sequences of BiP from the two
species revealed regions of sequence conservation (Fig. 4B).
Immunofluorescence with the anti-BiP antibody
Wild-type cells were fixed with formaldehyde and
processed for immunofluorescence using the anti-BiP antibody. The nuclear envelope, elements near the plasma
membrane and strands through the cytoplasm were labelled
(Fig. 5A). This distribution was also observed by confocal
microscopy of the mid-section of cells (Fig. 5B); in addition
images from above or below the mid-section revealed a
peripheral reticulum (Fig. 5C). Thus the distribution of
wild-type BiP is the same as that previously reported for
epitope-tagged protein (Pidoux and Armstrong, 1992).
The behaviour of the ER during the cell cycle, and particularly the nuclear envelope, could be followed using the
anti-BiP antibody (Fig. 6). The cell in Fig. 6A has just
divided from its sister cell; staining of the nuclear envelope
and peripheral ER is apparent. Cell growth occurs at the
ends of the cell (Fig. 6B, C) with cell length increasing
from 7 to 14 µm through the cell cycle. Early in anaphase,
when the chromosomes begin to separate, the nucleus elongates as is shown by the shape of the nuclear envelope staining in Fig. 6D. As the spindle elongates in anaphase B the
nuclei move apart (Fig. 6E, F). In Fig. 6E labelling that
appears to correspond to two thicknesses of nuclear envelope membrane is seen stretching between the nuclei. The
nuclei in fact move almost to the ends of the cell before
they return to the centre of the two incipient daughter cells
(Fig. 6G). By this time the nuclear envelope, which was
BiP from fission yeast 1119
Fig. 6. Confocal immunofluorescence of BiP in cells at different stages of the division cycle. The nuclear envelope elongates (B,C) until
the daughter nuclei are connected by a thick strand of BiP-labelled membrane (D-F). Just before and during cell division BiP protein
appears concentrated at the equator (G). Bar, 2 µm.
stretched between the two nuclei, has virtually disappeared.
There is a striking concentration of ER staining at the equator of cells that are about to divide or are dividing (Fig. 6E,
G). Further images of this phenomenon are shown in Fig.
7. The reticular structures are present around the cell periphery throughout the cell cycle and do not appear to break
down at any stage (data not shown).
DISCUSSION
The BiP protein is of interest for several reasons: its role
in folding and assembly of membrane and secretory proteins, its inducibility following a variety of cellular stresses,
and as a model protein retained in the ER lumen. Previously we reported the cloning of the BiP gene from the fission yeast S. pombe, and its unusual retention signal, ADEL
(Pidoux and Armstrong, 1992). Here we have described an
immunological analysis of the BiP protein, using an antibody raised to bacterially expressed C-terminal fragments
of the protein.
An unexpected feature of the predicted amino acid
sequence was a site for N-linked glycosylation. Such sites
are generally absent from BiP sequences; in contrast, they
are present but necessarily unused in cytoplasmic members
of the hsp70 family. In an epitope-tagged BiP the site was
used, but in only a small proportion of the molecules
(Pidoux and Armstrong, 1992). We have shown here that
the same applies to the natural protein, and in addition that
the extent of glycosylation does not increase with time, in
spite of the co-localisation of BiP with the glycosylation
machinery in the ER (Fig. 2). Thus the glycosylation site
presumably is inaccessible after the protein has been
translocated, folded and released into the ER lumen. One
possibility apparently eliminated by these results is that gly-
Fig. 7. Confocal immunofluorescence of BiP in cells undergoing
cytokinesis. Before cell division (A-E) there is a concentration of
labelling at the cell equator, appearing as a patch or spot. This
material appears to be shared between the two daughter cells upon
division (F). Bar, 2 µm.
cosylation is a late event related to degradation of the protein. Hence the functional significance of the partial glycosylation remains unknown.
1120 A. L. Pidoux and J. Armstrong
We investigated the ability of the antibody to react with
BiP from other species of yeast. Antibodies to conserved
luminal proteins of the secretory pathway in general show
quite a restricted specificity, thereby avoiding reaction with
the homologous protein of the host species within the ER
or Golgi of the antibody-producing cell. The antibody did
not react with mammalian cells (not shown), or with the
budding yeasts S. cerevisiae or Pichia pastoris (Fig. 4A).
Conversely, an antibody to BiP of S. cerevisiae (Rose et
al., 1989) did not cross-react with S. pombe (not shown).
The S. pombe antiserum did detect a protein in the related
fission yeast S. japonicus and also, surprisingly, in the budding yeast K. lactis (Fig. 4A). The sequence of BiP from
the latter species is known (Lewis and Pelham, 1990). Comparison of the sequences revealed a short region of sequence
conservation near the C terminus (Fig. 4B). Previously we
showed that the C-terminal sequence of S. pombe BiP,
ADEL, acted as an ER retention signal in this species, and
that variants from other species, including the K. lactis
sequence DDEL, could also function but less efficiently
(Pidoux and Armstrong, 1992). The conserved sequence
includes the first three of these residues, DDE, which precede the ADEL signal in the S. pombe protein. It may be
of interest to determine if other ER proteins from the two
species are similarly related at their C termini.
Our previous immunofluorescence analysis of epitopetagged BiP protein in S. pombe revealed, in addition to the
nuclear envelope, a polygonal reticular structure in the cell
periphery reminiscent of the reticulum of higher cells
(Pidoux and Armstrong, 1992). Immunofluorescence with
the anti-BiP antibody confirmed that this structure is constitutive and not an artefact of the expression system (Fig.
5C). During mitosis in higher cells the nuclear envelope
and, to some extent the peripheral ER, vesiculate and then
reassemble around the daughter nuclei. In contrast S.
pombe, like other yeasts, undergoes closed mitosis in which
the nuclear envelope elongates and then divides without
breaking down. We have observed the stages of this process
in S. pombe by immunofluorescence of BiP in cells at different points of the cell cycle (Fig. 6). A striking feature is
the appearance of a thick strand of material that connects
the two separating nuclei and then disappears before the
daughter cells separate (Fig. 6E-G). The fate of this membrane is unknown; it may shrink back around the two nuclei,
or form cytoplasmic ER, or be degraded. Alternatively, it
may contribute to the concentration of membrane that
appears at the equator (Figs 6G, 7).
The appearance of these structures raises numerous questions concerning their topology, function and coordination
with other events in the cell cycle. If the filamentous struc-
ture comprises two concentric tubules separately connected
to the inner and outer nuclear envelopes, do the separate
layers have different compositions reflecting their distinct
origins? Is the process of membrane breakage purely
mechanical, or does it require a distinct scission activity?
Is the ER material at the equator specifically involved in
generating the new plasma membrane that subsequently
forms at the same site? Future work may help to integrate
the answers to these questions with the wealth of information already available concerning other aspects of the cell
cycle in S. pombe.
We thank Jeremy Hyams and Kevin Hardwick for yeast strains,
Mark Rose for antibody, Vas Ponnambalam for fusion protein,
and Kathryn Ayscough, Sally Bowden, Mark Craighead, Kevin
Hardwick and Vas Ponnambalam for helpful discussions during
the course of this work.
REFERENCES
Armstrong, J., Ponnambalam, S., Craighead, M., Watson, R. and
Bowden, S. (1993). Schizosaccharomyces pombe ypt5: a homologue of
the rab5 endosome fusion regulator. Mol. Biol. Cell (in press).
Gething, M.-J. and Sambrook, J. (1992). Protein folding in the cell.
Nature 355, 33-45.
Haas, I. G. and Wabl, M. (1983). Immunoglobulin heavy chain binding
protein. Nature 306, 387-389.
Lewis, M. J. and Pelham, H. R. B. (1990). The sequence of the
Kluyveromyces lactis BiP gene. Nucl. Acids Res. 18, 6438.
Munro, S. and Pelham, H. R. B. (1986). An hsp70-like protein in the ER:
identity with the 78 kd glucose-regulated protein and immunoglobulin
heavy chain binding protein. Cell 46, 1094-1101.
Munro, S. and Pelham, H. R. B. (1987). A C-terminal signal prevents
secretion of luminal ER proteins. Cell 48, 899-907.
Normington, K., Kohno, K., Kozutsumi, Y., Gething, M.-J. and
Sambrook, J. (1989). S. cerevisiae encodes an essential protein
homologous in sequence and function to mammalian BiP. Cell 57, 12231236.
Pidoux, A. L. and Armstrong, J. (1992). Analysis of the BiP gene and
identification of an ER retention signal in Schizosaccharomyces pombe.
EMBO J. 11, 1583-1591.
Rose, M. D., Misra, L. M. and Vogel, J. P. (1989). KAR2, a karyogamy
gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell 57,
1211-1221.
Smith, D. B. and Johnson, K. S. (1988). Single-step purification of
polypeptides expressed in Escherichia coli as fusions with glutathione-Stransferase. Gene 67, 31-40.
Spindler, K. R., Rosser, D. S. E. and Berk, A. J. (1984). Analysis of
adenovirus transforming proteins from early regions 1A and 1B with
antisera to inducible fusion antigens in E. coli. J. Virol. 49, 132-141.
Vogel, P., Misra, L. M. and Rose, M. D. (1990). Loss of BiP/GRP78
function blocks translocation of secretory proteins in yeast. J. Cell Biol.
110, 1885-1895.
(Received 22 October 1992 - Accepted, in revised form,
27 April 1993)